Lecture 7: Mitochondrial DNA Profiling
Mitochondrial DNA and forensics Human mitochondrial genome Polymorphic regions DNA sequencing Interpretation of Results Identifying Tsar Nicholas and family The mystery of Anna Anderson 2
DNA found in mitochondria Bacteria-like Circular No recombination Short and “no-nonsense” Main advantages in forensics: More copies in cell; less subject to degradation ▪ Hair shaft ▪ Bones ▪ Decomposed samples 3
Main disadvantages in forensics Low power of discrimination ▪ Maternally inherited ▪ No recombination Less polymorphic than nuclear DNA ▪ Most common Caucasian type found in 7.1% of all Caucasians More subject to contamination during analysis than nuclear DNA ▪ More copies per cell – including cells from analyst or other sources 4
Mitochondria = Sub-cellular organelles which are generated ATP from breakdown of food 5
16,569 nucleotides (more or less) and 37 genes 13 genes coding for proteins in ETC 24 genes coding for tRNA and rRNA A person’s mtDNA sequence is called a mitotype 10x higher mutation rate than nuclear genome Revised Cambridge Reference Sequence First human mtDNA genome sequenced Later revised to correct mistakes: rCRS Used as reference for mitotype nomenclature 6
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Located in control region called D-Loop No genes in D-loop Hypervariable regions: HV1 (16,024-16,365; 342 bp) HV2 (73-340; 248 bp) HV3 ( ; 137 bp) Most commonly region in forensics: ▪ HV1 ▪ HV2 8
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Mitotype reporting Reported against rCRS Sequence polymorphisms; requires DNA sequencing ▪ E.g T; 73A Length polymorphisms ▪ Insertions or deletions ▪ Insertions: E.g A, 524.2C ▪ Deletions: E.g d Heteroplasmy More than one detectable mitotype in a person or some tissues in a person (esp. hair)
Current method developed by Sanger Uses modified nucleotides called ddNTPS ▪ Dideoxynucleotide triphosphates (ddATP, ddGTP, ddTTP, ddCTP) 11
Reaction contains: Denatured “template” DNA Short, synthetic single-stranded DNA primer Large concentration of dNTPs (normal nucleotide triphosphates) Small concentration of ddNTPs ▪ Labeled with different fluorescent dyes (e.g. G black, A green, T red, C purple) DNA polymerase Salts and buffers 12
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14 Chromas Lite CODISmt
Scientific working group on DNA analysis methods (SWGDAM) and International Society of Forensic Genetics (ISFG) Cannot exclude ▪ Questioned sample has same sequence as reference sample Exclusion ▪ Two or more nucleotides differences between questioned sample and reference sample Inconclusive ▪ Questioned and reference sample differ by one nucleotide 15
Russian Tsar Nicholas II and family removed from power and murdered during Bolshevik Revolution in 1918 Shot by firing squad, doused with sulfuric acid, buried in a shallow pit under a road Remains went undiscovered until 1991 ▪ Nine skeletons discovered (4 male adults, 2 female adults, and 3 female children) ▪ Unrecognizable by any method other than DNA ▪ Too decomposed for nuclear DNA typing (RFLP) 16
mtDNA extracted from femur of each skeleton Blood samples obtained from maternally- related descendants Tsarina Alexandra ▪ Prince Phillip (England) is grand nephew of unbroken maternal descent ▪ His sequence matched that of one adult female skeleton (the Tsarina) and all 3 female children’s skeletons Tsar ▪ Sequence of adult male skeletons compared to two relatives of unbroken maternal descent to Tsar 17
Single nucleotide difference found at 16,169 ▪ Tsar had heteroplasmy at this site (T and C detected in sequencing reaction) ▪ Putative relatives had only T ▪ Inconclusive Body of Tsar’s brother exhumed and tested ▪ Tests showed same heteroplasmy as Tsar ▪ Tsar’s identity confirmed ▪ Eye-witness reports indicate that the footman, the family cook, and the family doctor were the other 3males in the grave; the Tsarina’s assistant was the second adult female 18
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Tsar had four daughters and one son ▪ One daughter and the son not accounted for In 2007, two additional bodies found in same area ▪ mtDNA testing confirmed that they belonged to the missing daughter and son 20